LOCUS       sequence-133555-        5557 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  C-terminal tagging with mCherry2 using CRIPSR/Cas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5557)
  AUTHORS   Natsume T, Kiyomitsu T, Saga Y, Kanemaki MT
  TITLE     Rapid Protein Depletion in Human Cells by Auxin-Inducible Degron 
            Tagging with Short Homology Donors.
  JOURNAL   Cell Rep. 2016 Apr 5;15(1):210-8. doi: 10.1016/j.celrep.2016.03.001.
            Epub 2016 Mar 24.
  PUBMED    27052166
REFERENCE   2  (bases 1 to 5557)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2016.03.001"; journalName: "Cell Rep"; date: 
            "2016-04-5- 5"; volume: "15"; issue: "1"; pages: "210-8"
COMMENT     Sequence Label: pMK282 (mCherry2-Bsr)
FEATURES             Location/Qualifiers
     source          1..5557
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..459
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(91..110)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     301..322
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             683..1393
                     /codon_start=1
                     /product="derivative of the monomeric red fluorescent 
                     protein mCherry"
                     /label=mCherry2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQCRTMGWEASTERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(828..846)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     polyA_signal    1511..1632
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1548..1567)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1602..1621
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    1663..1696
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        1757..2256
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1792..1815)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2189..2208
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     protein_bind    3287..3320
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(3369..3389)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3369..3387)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3408..3424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3408..3424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3421..3443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3432..3448
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3456..3462,3463..3480,3481..3486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3501..3522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3639..3656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3810..4398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3890..3909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4569..5360,5361..5429))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     5192..5211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5430..5534)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga
       61 ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg
      121 ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat
      181 ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg
      241 ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
      301 gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt
      361 tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat
      421 ttaacgcgaa ttttaacaaa atattaacgc ttacaatttc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tggagctcgg
      661 atccggtgca ggcgccgcta gcatggtgag caagggcgag gaggataaca tggccatcat
      721 caaggagttc atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga
      781 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
      841 gaccaagggt ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg
      901 ctccaaggcc tacgtgaagc accccgccga catccccgac tacttgaagc tgtccttccc
      961 cgagggcttc aattgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac
     1021 ccaggactcc tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa
     1081 cttcccctcc gacggccccg taatgcagtg tcgtaccatg ggctgggagg cctccactga
     1141 gcggatgtac cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa
     1201 ggacggcggc cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca
     1261 gctgcccggc gcctacaacg tcgacatcaa gttggacatc ctttcccaca acgaggacta
     1321 caccatcgtg gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga
     1381 gctgtacaag taatctagat aactgatcat aatcagccat accacatttg tagaggtttt
     1441 acttgcttta aaaaacctcc cacacctccc cctgaacctg aaacataaaa tgaatgcaat
     1501 tgttgttgtt aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     1561 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     1621 caatgtatct taacgcgtcg atcatattca ataaccctta atataacttc gtataatgta
     1681 tgctatacga agttattagg tctgaagagg agtttacgtc cagccaagct taggatctcg
     1741 acctcgaaat tctaccgggt aggggaggcg cttttcccaa ggcagtctgg agcatgcgct
     1801 ttagcagccc cgctgggcac ttggcgctac acaagtggcc tctggcctcg cacacattcc
     1861 acatccaccg gtaggcgcca accgactccg ttctttggtg gccccttcgc gccaccttct
     1921 actcctcccc tagtcaggaa gttccccccc gccccgcagc tcgcgtcgtg caggacgtga
     1981 caaatggaag tagcacgtct cactagtctc gtgcagatgg acagcaccgc tgagcaatgg
     2041 aagcgggtag gcctttgggg cagcggccaa tagcagcttt gctccttcgc tttctgggct
     2101 cagaggctgg gaaggggtgg gtccgggggc gggctcaggg gcgggctcag gggcggggcg
     2161 ggcgcccgaa ggtcctccgg aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc
     2221 gcgctgttct cctcttcctc atctccgggc ctttcgacct gcatccatct agatctcgag
     2281 cagctgaagc ttaccgctag catgaaaaca tttaacattt ctcaacaaga tctagaatta
     2341 gtagaagtag cgacagagaa gattacaatg ctttatgagg ataataaaca tcatgtggga
     2401 gcggcaattc gtacgaaaac aggagaaatc atttcggcag tacatattga agcgtatata
     2461 ggacgagtaa ctgtttgtgc agaagccatt gcgattggta gtgcagtttc gaatggacaa
     2521 aaggattttg acacgattgt agctgttaga cacccttatt ctgacgaagt agatagaagt
     2581 attcgagtgg taagtccttg tggtatgtgt agggagttga tttcagacta tgcaccagat
     2641 tgttttgtgt taatagaaat gaatggcaag ttagtcaaaa ctacgattga agaactcatt
     2701 ccactcaaat atacccgaaa ttaagctagc cgcccgcccc acgacccgca gcgcccgacc
     2761 gaaaggagcg cacgacccca tgcatcgatg atatcagatc cccgggatgc agaaattgat
     2821 gatctattaa acaataaaga tgtccactaa aatggaagtt tttcctgtca tactttgtta
     2881 agaagggtga gaacagagta cctacatttt gaatggaagg attggagcta cgggggtggg
     2941 ggtggggtgg gattagataa atgcctgctc tttactgaag gctctttact attgctttat
     3001 gataatgttt catagttgga tatcataatt taaacaagca aaaccaaatt aagggccagc
     3061 tcattcctcc cactcatgat ctatagatct atagatctct cgtgggatca ttgtttttct
     3121 cttgattccc actttgtggt tctaagtact gtggtttcca aatgtgtcag tttcatagcc
     3181 tgaagaacga gatcagcagc ctctgttcca catacacttc attctcagta ttgttttgcc
     3241 aagttctaat tccatcagaa gctggtcgag atccggaacc cttaatataa cttcgtataa
     3301 tgtatgctat acgaagttat taggtccctc gaagaggttc actaggatcc ggtacccagc
     3361 ttttgttccc tttagtgagg gttaattgcg cgcttggcgt aatcatggtc atagctgttt
     3421 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     3481 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     3541 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3601 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     3661 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     3721 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     3781 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     3841 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     3901 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     3961 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4021 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4081 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4141 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4201 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt
     4261 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4321 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4381 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4441 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     4501 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     4561 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     4621 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     4681 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     4741 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     4801 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     4861 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     4921 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     4981 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5041 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5101 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5161 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5221 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5281 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5341 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5401 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     5461 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     5521 ataggggttc cgcgcacatt tccccgaaaa gtgccac
//
